Process for the enzymatic preparation of an amide from a nitrile

ABSTRACT

Disclosed herein are cyanide-tolerant nitrile hydratases especially from  Pseudomonas putida  or  Pseudomonas marginalis  strains which exhibit increased cyanide tolerance. Also disclosed are methods of preparing amides from nitriles in the presence of cyanides and polynucleotide sequences coding for cyanide-tolerant nitrile hydratases.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to cyanide-tolerant nitrile hydratases especiallyfrom Pseudomonas putida or Pseudomonas marginalis strains which exhibitincreased cyanide tolerance, to their use for preparing amides fromnitrites in the presence of cyanides and to polynucleotide sequencescoding for this enzyme.

2. Description of the Related Art

The conversion of α-hydroxy nitrites (cyanohydrins) and α-amino nitritesinto the corresponding amides using nitrile hydratases opens up a novelvariant for synthesizing α-hydroxy acids and α-amino acids becauseα-hydroxy amides and α-amino amides can be hydrolyzed in a simple manner(Process and catalysts for the production of methionine. Ponceblanc,Herve; Rossi, Jean-Christophe; Laval, Philip; Gros, Georges.(Rhone-Poulenc Animal Nutrition SA, Fr.), (WO 2001060789).Alternatively, α-hydroxy amides can also be reacted with alkali metal oralkaline earth metal hydroxides to give the corresponding salts of thehydroxy acids. A particularly preferred reaction in this connection isthat of 4-methylthio-α-hydroxybutyramide (MHA amide) with calciumhydroxide, because calcium MHA can be employed directly as alternativeform of product to methionine or MHA as feed additive.

However, α-hydroxy nitrites and α-amino nitrites readily decompose toaldehydes and hydrocyanic acid, and aldehydes, hydrocyanic acid andammonia, respectively. The resulting hydrocyanic acid is a stronginhibitor of almost all known nitrile hydratases with the exception ofthe nitrile hydratase from Rhodococcus equi XL-1, which shows thesmallest loss of activity known to date at 20 mM cyanide (Production ofamides from nitrites by Rhodococcus equi cells having a cyanideresistant-nitrile hydratase. Nagasawa, Tohru; Matsuyama, Akinobu.(Daicel Chemical Industries, Ltd., Japan), (EP 1 266 962 A).

The low productivity of about 8 g of amide per g of dry biomass ofresting cells, the long reaction time of 43 hours and the relatively lowproduct concentration of 75 g/l lead to the search for improved nitrilehydratases.

SUMMARY OF THE INVENTION

The aim of the invention described herein is therefore to provide abiocatalyst which is not subject to these restrictions. In addition, aneven greater tolerance of cyanide by the biocatalyst is advantageous,because α-hydroxy nitrites and α-amino nitrites are prepared, in orderto ensure a rapid and complete reaction of the aldehyde, preferably witha 1-3% excess of hydrocyanic acid, part of which remains in the product.It is thus possible for cyanide concentrations exceeding 20 mM to occurduring the biotransformation. By-products and reagents such as aminesemployed as auxiliary bases must likewise not inhibit the nitrilehydratase activity.

It is an object of the invention to provide nitrile hydratases whichexhibit an increased stability to the cyanide ions present in thereaction solution during the conversion of nitrites to amides.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the relative activity of strain MA32.

FIG. 2 is a graph showing the relative activity of strain MA113.

FIG. 3 is a graph showing the relative activities for the conversion ofmethacrylonitrile as a function of the cyanide concentration of strainMA32.

FIG. 4 is a graph showing the relative activities for the conversion ofmethacrylonitrile as a function of the cyanide concentration of strainMA113.

FIG. 5 shows the time course of conversion of acetone cyanohydrinachieved with strain MA113.

FIG. 6 shows the time course of conversion of crude MHA nitrile achievedwith strain MA32.

FIG. 7 schematically shows the restriction map of the expression vectorpKE31.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to isolated polynucleotides, in particular frommicroorganisms of the genus Pseudomonas, which code for polypeptideshaving the amino acid sequences which are 90 to 100% identical to theamino acid sequences comprised in the sequences SEQ ID NO: 2, 3, 5, 7,8, 10, where the polypeptides comprising the sequences SEQ ID NO: 2, 3,5 or 7, 8, 10, together in each case have the activity of acyanide-tolerant nitrile hydratase or form this nitrile hydratase.

The polynucleotides are preferably derived from Pseudomonas putida orPseudomonas marginalis.

The invention further relates to polynucleotides selected from the groupof

-   a) polynucleotides comprising or consisting of the nucleotide    sequences from SEQ ID NO: 1, 4, 6, 9 or nucleotide sequences    complementary thereto,-   b) polynucleotides comprising nucleotide sequences which correspond    to the sequences from a) within the scope of the degeneracy of the    genetic code,-   c) polynucleotides comprising nucleotide sequences as in a) which    comprise functionally neutral sense mutations,-   d) polynucleotides which hybridize with the complementary sequences    from a) or c) under stringent conditions,    where the polynucleotides code for a cyanide-tolerant nitrile    hydratase.

The invention likewise relates to the polypeptides encoded by thesepolynucleotides and having the sequences SEQ ID NO: 2, 3, 5 or 7, 8, 10with the activity of cyanide-tolerant nitrile hydratases frommicroorganisms of the genus Pseudomonas, which may be present eitherenriched in the microorganisms or in isolated form. SEQ ID NO: 2 and 7code for the alpha subunits of the nitrile hydratases, SEQ ID NO: 3 and8 for the beta subunits of the nitrile hydratases and SEQ ID NO: 5 and10 for activator proteins whose coexpression is essential for theactivity of the nitrile hydratases (Nojiri et al., 1999, Journal ofBiochemistry, 125: 696-704).

It is preferred according to the invention to use host cells which havebeen transformed or transfected by the polynucleotides of the invention.

The host cells may belong to the eukaryotes or prokaryotes for which astable expression system is known, in particular

the host organisms preferably used are microorganisms for which thereare expression systems, such as, for example, Pseudomonas, Pichia,various yeasts, Saccharomyces, Aspergillus or the Streptomyces family,especially E. coli. Microorganisms of the genus Rhodococcus are likewisesuitable.

Vector DNA can be introduced into eukaryotic or prokaryotic cells byknown techniques of transformation or transfection.

“Transformation”, “transfection”, conjugation” and “transduction” referto procedures known in the state of the arts for introducing foreignDNA.

The invention likewise relates to polynucleotides which consistessentially of one polynucleotide sequence, which are obtainable byscreening by means of hybridization of an appropriate gene library ofPseudomonas marginalis or Pseudomonas putida which comprises thecomplete gene or parts thereof, with a probe which comprises thesequences of the polynucleotides of the invention from SEQ ID No: 1, 4or 6, 9 or fragments thereof, and isolation of said polynucleotidesequence.

Polynucleotides which comprise the sequences of the invention aresuitable as hybridization probes for RNA, cDNA and DNA in order toisolate full-length nucleic acids and polynucleotides or genes whichcode for the proteins of the invention, or in order to isolate thosenucleic acids and polynucleotides or genes which exhibit a greatsimilarity of the sequences to those of the genes of the invention. Theycan likewise be attached as probe to so-called arrays, microarrays orDNA chips in order to detect and determine the correspondingpolynucleotides or sequences derived therefrom, such as, for example,RNA or cDNA.

Polynucleotides which comprise the sequences of the invention arefurther suitable as primers with whose aid it is possible with thepolymerase chain reaction (PCR) to prepare DNA of genes which code forthe proteins of the invention.

Such oligonucleotides serving as probes or primers comprise at least 25or 30, preferably at least 20, very particularly preferably at least 15,consecutive nucleotides. Oligonucleotides having a length of at least 40or 50 nucleotides are likewise suitable. Also suitable where appropriateare oligonucleotides having a length of at least 100, 150, 200, 250 or300 nucleotides.

“Isolated” means separated from its natural environment.

“Polynucleotide” refers in general to polyribonucleotides andpolydeoxyribonucleotides, possibilities being unmodified RNA or DNA ormodified RNA or DNA.

The polynucleotides of the invention include polynucleotides of SEQ IDNo: 1, 4, 6, 9 or fragments contained therein, and also those which areat least 90%, 93%, 95%, 97% or 99% identical to the polynucleotides ofSEQ ID NO: 1, 4, 6, 9 or fragments contained therein.

“Polynucleotides” mean peptides or proteins which comprise two or moreamino acids connected by peptide linkages.

The polypeptides of the invention include polypeptides of sequences SEQID NO: 2, 3, 5, 7, 8, 10, and also those which are at least 90%, andparticularly preferably at least 91%, 95%, 97% or 99% identical to thepolypeptides of sequences SEQ ID NO: 2, 3, 5, 7, 8, 10.

The DNA sequences obtained from the desired gene library can then beexamined using known algorithms or sequence analysis programs such as,for example, that of Staden (Nucleic Acids Research 14, 217-232 (1986)),that of Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or the GCGprogram of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).

Coding DNA sequences which are derived from the sequences comprised inSEQ ID No. 1, 4, 6, 9 through the degeneracy of the genetic code arelikewise an aspect of the invention. In the same way, DNA sequenceswhich hybridize with the sequences or parts thereof are an aspect of theinvention. Also known to experts are conservative amino acid exchangessuch as, for example, exchange of glycine for alanine or of asparticacid for glutamic acid in proteins as “sense mutations” which do notlead to a fundamental change in the activity of the protein, i.e. arefunctionally neutral. It is further known that changes at the N or Cterminus of a protein may not substantially impair or even stabilize itsfunction. Details concerning this are to be found by a person skilled inthe art inter alia in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77: 237-251 (1989)), inSahin-Toth et al. (Protein Sciences 3: 240-247 (1994)), in Hochuli etal. (Bio/Technology 6: 1321-1325 (1988)) and in well-known textbooks ofgenetics and molecular biology.

Finally, DNA sequences which are prepared by the polymerase chainreaction (PCR) using primers which are derived from SEQ ID NO: 1, 4, 6,9 are an aspect of the invention. Such oligonucleotides typically have alength of at least 15 consecutive nucleotides, in particular of 20, 30or 40.

Instructions for the identification of DNA sequences by means ofhybridization are to be found by a person skilled in the art inter aliain the handbook “The DIG System Users Guide for Filter Hybridization” ofBoehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al.(International Journal of Systematic Bacteriology (1991) 41: 255-260).The hybridization takes place under stringent conditions, meaning thatonly hybrids in which probe and target sequence, i.e. thepolynucleotides treated with the probe, are at least 90% identical areformed. It is known that the stringency of the hybridization includingthe washing steps is influenced or determined by variation in the buffercomposition, the temperature and the salt concentration. Thehybridization reaction is preferably carried out with relatively lowstringency compared with the washing steps (Hybaid Hybridisation Guide,Hybaid Limited, Teddington, UK, 1996).

It is possible to employ for the hybridization reaction for example a5×SSC buffer at a temperature of about 50° C.-68° C. It is possible inthis case also for probes to hybridize with polynucleotides exhibitingless than 70% identity to the sequence of the probe. Such hybrids areless stable and are removed by washing under stringent conditions. Thiscan be achieved for example by lowering the salt concentration to 2×SSCand, where appropriate, subsequently 0.5×SSC (The DIG System User'sGuide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany,1995), setting a temperature of about 50° C.-68° C. It is possible whereappropriate to lower the salt concentration to a 0.1×SSC. It is possibleby raising the hybridization temperature stepwise in steps of about 1-2°C. from 50° C. to 68° C. to isolate polynucleotide fragments which have,for example, at least 90% to 95% identity to the sequence of the probeemployed. Further instructions for hybridization are obtainable on themarket in the form of so-called kits (e.g. DIG Easy Hyb from RocheDiagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558).

Instructions for the amplification of DNA sequences with the aid of thepolymerase chain reaction (PCR) are to be found by a person skilled inthe art inter alia in the handbook by Gait: Oligonukleotide synthesis: APractical Approach (IRL Press, Oxford, UK, 1984) and in Newton andGraham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

The procedure is generally such that a gene which can be expressed wellis cloned into a vector with lower copy number, genes with lowerexpression efficiency on a vector with higher copy number and/or strongpromoter. The host cells are transformed with these vectors in such away that, compared with the initial organism, they comprise at least ineach case one additional copy of the nucleotide sequences coding for theformation of nitrile hydratase.

The transformed or recombinant microorganisms prepared in this way,especially of the genus Pseudomonas, are likewise part of the invention.

It has been found that enhancement of the genes coding for the nitrilhydratase of the invention and the helper protein P47K in microorganismsleads to an increased production of the nitrile hydratase or else to anincreased activity of the nitrile hydratase.

The term “enhancement” describes in this connection the increase in theintracellular activity of one or more enzymes in a microorganism whichare encoded by the appropriate DNA by, for example, increasing the copynumber of the gene or of the genes, using a strong promoter or using agene which codes for a corresponding enzyme with a high activity and,where appropriate, combining these measures, compared with thenon-recombinant initial organism.

To achieve overexpression it is possible to mutate the promoter andregulatory region or the ribosome binding site which is located upstreamof the structural gene. Expression cassettes incorporated upstream ofthe structural gene work in the same way. It is additionally possible toincrease expression during the fermentative amino acid production byinducible promoters. Expression is likewise improved by measures toextend the lifespan of the m-RNA.

In addition, the enzymatic activity is likewise enhanced by preventingdegradation of the enzyme protein. The genes or gene constructs may bepresent either in plasmids with varying copy number or integrated andamplified in the chromosome. A further alternative possibility is toachieve overexpression of the relevant genes by modifying thecomposition of the media and management of the culturing.

The invention likewise relates to

-   1) a process for the enzymatic preparation of amides from nitrites,    which comprises the following steps:    -   a) conversion of a compound which comprises a nitrile group or        nitrile groups using a microbial enzyme which has nitrile        hydratase activity and    -   b) removal of the amide formed, where    -   c) a nitrile hydratase of the invention is employed for        converting the nitrile into the amide. The remaining activity        thereof after conversion of methacrylonitrile in the presence of        20 mM (mM=mmol/l) cyanide ions at 20° C. after 30 min is        preferably at least 90% of the remaining activity of the same        enzyme when it has been employed for the conversion in the        absence of cyanide ions under conditions which are otherwise the        same.-   2) a process according to 1), characterized in that the remaining    activity after the conversion in the presence of 50 mM cyanide ions    is at least 60%,-   3) a process according to 1) or 2), characterized in that    microorganisms producing and containing enzyme, or the lysate    thereof, is/are employed.-   4) a process according to 3), characterized in that resting cell of    the microorganism is employed,-   5) a process according to 1) or 2), characterized in that the    purified enzyme is employed,-   6) a process according to 1) to 5), characterized in that the enzyme    is derived from microorganisms of the genus Pseudomonas, in    particular Pseudomonas putida or Pseudomonas marginalis,-   7) a process according to 6, characterized in that the enzyme is    derived from microorganisms of the genus Pseudomonas deposited under    the numbers DSM 16275 and DSM 16276, and which have amino acid    sequences having the sequences SEQ ID NO: 2, 3, 5, 7, 8, 10,-   8) a process according to one or more of points 1) to 7),    characterized in that compounds of the general formulae

-   -   in which the meanings are:    -   X: OH, H, alkyl having 1 to 4 C atoms, NH₂    -   R: H, saturated alkyl radical having 1 to 12 C atoms, branched        or unbranched, optionally NH₂-substituted        -   unsaturated alkyl radicals having a double bond and 1 to 12            C atoms, branched or unbranched, cycloalkyl groups having 3            to 6 C atoms,        -   alkylene radicals substituted by alkylthio groups, where            alkyl here corresponds to a C₁ to C₃ radical,        -   and alkylene corresponds to a divalent C₃ to C₈ radical,    -   R′: H, if R is not H, alkyl having 1 to 3 C atoms,    -   R″: mono- or binuclear unsaturated ring having 6 to 12 C atoms,        optionally substituted by one or two alkyl groups (C₁-C₃), Cl,        Br, F, monovalent alkyl nitrile radical having 1 to 6 C atoms,    -   are converted to the corresponding amides,

-   9) a process according to 8), characterized in that a compound of    the general formula (I) is converted in the presence of hydrocyanic    acid or a salt of hydrocyanic acid,

-   10) a process according to 9), characterized in that the conversion    is carried out in the presence of 0.1 mol % cyanide to 3 mol %    cyanide based on the nitrile employed, preferably >2 to 3 mol %.    This corresponds at a final concentration of 1 mol to 30 mmol of    cyanide at 3 mol %,

-   11) a process according to one or more of points 1) to 10),    characterized in that methionine nitrile is employed as nitrile,

-   12) a process according to one or more of points 1) to 10),    characterized in that 2-hydroxy-4-methyl-thiobutyronitrile is    employed as nitrile.    -   A reaction mixture like that obtained when hydrocyanic acid,        3-methylthiopropionaldehyde are reacted in the presence of an        auxiliary base such as, for example, triethylamine according to        the prior art is preferably employed.    -   It can advantageously be employed without purification.    -   This indicates the additional stability of the enzymes of the        invention toward aldehydes and amines.

-   13) A process in which 2-hydroxy-2-methylpropionitrile is employed    as precursor for methacrylamide.

-   14) The invention is likewise directed to isolated and purified    microorganisms of the genus Pseudomonas, deposited under the numbers    DSM 16275 (MA32, Pseudomonas marginalis) and DSM 16276 (MA113,    Pseudomonas putida), and

-   15) cyanide-tolerant nitrile hydratases isolated from the strains of    the genus Pseudomonas, in particular from the strains of Pseudomonas    putida and Pseudomonas marginalis deposited under the numbers DSM    16275 and DSM 16276.

The deposition took place on Mar. 9, 2004, at the DSMZ, DeutscheSammlung für Mikroorganismen und Zellkulturen in Brunswick, inaccordance with the Budapest treaty.

The strains are particularly suitable for producing the enzymes of theinvention.

“Isolated and purified microorganisms” relates to microorganisms whichare present in a higher concentration than found naturally.

The invention likewise relates to a process for preparing thecyanide-tolerant nitrile hydratase described above, in which

-   a) a microorganism producing this nitrile hydratase, in particular    of the genus Pseudomonas marginalis or Pseudomonas putida, is    fermented under conditions with which the enzyme is formed in the    microorganism, and-   b) the cells are harvested at the earliest after the logarithmic    growth phase has been completed.

Subsequently,

-   a) either the microorganism comprising the enzyme in the form of    resting cells, where appropriate after increasing the permeability    of the cell membrane, or-   b) the lysate of the cells or-   c) the enzyme isolated from the cells of the microorganism using    known measures    is employed for the conversion according to the invention of    nitrites into amides.

The nitrile hydratase may be either an enzyme generated withnon-recombinant microorganisms or an enzyme generated recombinantly.

The invention additionally relates to processes for the recombinantpreparation of the polypeptides of the invention, where a microorganismproducing these polypeptides is cultivated, where appropriate expressionof the relevant polynucleotides is induced, and the enzymes are isolatedwhere appropriate from the culture.

The process is generally one in which

-   a) microorganisms in particular of the genera Pseudomonas marginalis    or Pseudomonas putida in which isolated polynucleotides from    microorganisms of the family Pseudomonas which code for polypeptides    having the amino acid sequences which are 90 to 100% identical to    the amino acid sequences comprising sequences in SEQ ID NO: 2, 3 and    5 or 7, 8 and 10, where the polypeptides in each case jointly have    the activity of a cyanide-tolerant nitrile hydratase, enhanced, in    particular recombinantly overexpressed, are fermented,-   b) the enzyme having nitrile hydratase activity is isolated where    appropriate from these microorganisms, or a protein fraction    comprising this enzyme is prepared, and-   c) the microorganism according to a) or the enzyme according to or    the fraction comprising the latter b) is transferred into a medium    which comprises a compound comprising nitrile groups of the general    formulae (I) and (II).

The culture medium used for the fermentation must comply in a suitablemanner with the demands of the respective strains. Descriptions ofculture media of various microorganisms are present in the handbook“Manual of Methods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

It is possible to use as carbon source sugars and carbohydrates such as,for example, glucose, sucrose, lactose, fructose, maltose, molasses,starch and cellulose, oils and fats such as, for example, soybean oil,sunflower oil, peanut oil and coconut fat, fatty acids such as, forexample palmitic acid, stearic acid and linoleic acid, alcohols such as,for example, glycerol and ethanol and organic acids such as, forexample, acetic acid. These substances can be used singly or as mixture.

It is possible advantageously to use as nitrogen source organic nitritesor amides such as acetonitrile, acetamide, methacrylonitriles,methacrylamide, isobutyronitrile, isobutyramide or urea also incombination with other nitrogen-containing compounds such as peptones,yeast extract, meat extract, malt extract, corn steep liquor, soybeanflour and or inorganic compounds such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.The nitrogen sources may be used singly or as mixture.

It is possible to use as phosphorus source phosphoric acid, potassiumdihydrogen phosphate or dipotassium hydrogen phosphate or thecorresponding sodium-containing salts. The culture medium mustadditionally comprise salts of metals, such as, for example, magnesiumsulfate or iron sulfate, which are necessary for growth. Finally,essential growth factors such as amino acids and vitamins can beemployed in addition to the abovementioned substances. Said startingmaterials can be added to the culture in the form of a single batch orbe fed in during the cultivation in a suitable manner.

The pH of the culture is controlled in a suitable manner by employingbasic compounds such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia or acidic compounds such as phosphoric acid orsulfuric acid. Foaming can be controlled by employing antifoams such as,for example, fatty acid polyglycol esters. Aerobic conditions aremaintained by introducing oxygen or oxygen-containing gas mixtures suchas, for example, air into the culture. The temperature of the culture isnormally 10° C. to 40° C. and preferably 10° C. to 30° C. The culture iscontinued until it has passed through the logarithmic growth phase. Thisaim is normally achieved within 10 hours to 70 hours. Following this,the cells are preferably harvested, washed and taken up in a buffer assuspension at a pH of 6-9, in particular of 6.8 to 7.9. The cellconcentration amounts to 1-25%, in particular 1.5 to 15% (wet weight/v).The permeability can be increased by physical or chemical methods, e.g.with toluene as described in Wilms et al., J. Biotechnol., Vol. 86(2001), 19-30, so that the nitrile to be converted can penetrate throughthe cell wall and the amide can emerge.

The following nitrites are preferably converted:

Saturated Mononitriles:

acetonitrile, propionitrile, butyronitrile, isobutyronitrile,valeronitrile, isovaleronitrile, capronitrile

Saturated Dinitriles:

malonitrile, succinonitrile, glutaronitrile, adiponitrile

Aromatic Unsubstituted and Substituted Mono- and Dinitriles:

benzonitrile, 2,6-difluorobenzonitrile, phthalonitrile,isophthalonitrile, terephthalonitrile,

α-Amino Nitriles:

α-aminopropionitrile, α-aminomethylthiobutyronitrile,α-aminobutyronitrile, aminoacetonitrile, all nitriles derived fromnatural amino acids, α-amino-3,3-dimethyl-propionitrile,α-amino-2,3-dimethylpropionitrile

Nitriles with Carboxyl Groups:

cyanoacetic acid

β-Amino Nitriles:

3-aminopropionitril

Unsaturated Nitriles:

acrylonitrile, methacrylonitrile, allyl cyanide, crotononitrile

α-Hydroxy Nitriles:

α-hydroxy-n-propionitrile, α-hydroxy-n-butyronitrile,α-hydroxyisobutyronitrile, α-hydroxy-n-hexanonitrile,α-hydroxy-n-heptanonitrile, α-hydroxy-n-octanonitrile,α,γ-dihydroxy-β,β-dimethylbutyronitrile, acrolein cyano-hydrin,methacrylaldehyde cyanohydrin, 3-chloro-lactonitrile,4-methylthio-α-hydroxybutyronitrile and α-hydroxyphenylpropionitrile.

The concentration of the nitriles to be converted in the reactionsolution is not limited to particular ranges.

In order to avoid inhibition of the enzymatic activity via thesubstrate, the concentration of the nitrile is generally maintained at0.02 to 10 w/w %, in particular 0.1 to 2 w/w %, based on the amount ofthe biocatalyst as dry biomass. The substrate can be added as a whole atthe start of the conversion or continuously or discontinuously duringthe conversion.

The dry weight is determined using the Moisture Analyser MA 45(Sartorius).

If the solubility of the nitrile compound in the aqueous reaction systemis too low, a solubilizer can be added.

The reaction may, however, alternatively also be carried out in awater/organic solvent two-phase system.

When the cells of the microorganism are used as enzymatically activematerial, the amount of the cells employed is preferably 0.02 to 10 w/w% as dried biomass in relation to the amount of substrate.

It is also possible for the isolated enzyme to be immobilized bygenerally known techniques and then to be employed in this form.

The reaction is generally carried out at temperatures from −5° C. to 50°C., in particular 0° C. to 30° C., and for a time of from 0.1 to 100hours.

The pH of the reaction mixture which is to be maintained is not limitedto particular values as long as the enzymatic activity is not impaired.After the conversion, the amide formed can be removed from the reactionsolution as known and be purified.

The invention likewise relates to a process in which the amide or thesolution comprising the amide is separated for example from the cells ofthe biomass, and the amide is either hydrolyzed to the correspondingacid or converted with addition of alkali metal or alkaline earth metalhydroxides to the corresponding salts of the acids. MHA amide ispreferably hydrolyzed with calcium hydroxide and the correspondingcalcium salt is isolated.

EXAMPLES Example 1 Culturing Conditions

The precultures were grown in a volume of 5 ml in glass tubes, shakingat 30° C. over the course of 24 h. 100 ml of the main culture wereinoculated with 1 ml of the preculture and shaken in an Erlenmeyer flaskwith a total volume of 1000 ml at 25° C. for 42 h.

Medium for the preculture (pH 7.0) K₂HPO₄ 7 g KH₂PO₄ 3 g Na citrate 0.5g Glycerol 2 g FeSO4 * 7H₂O 0.004 g MgSO4 * 7H₂O 0.1 g Acetamide 2 gTrace salt solution 0.1 ml Demineralized water Ad.1000 ml Medium for themain culture (pH 7.0) K₂HPO₄ 7 g KH₂PO₄ 3 g Sodium citrate 0.5 gGlycerol 2 g FeSO4 * 7H₂O 0.004 g MgSO4 * 7H₂O 0.1 g Acetamide 10 gTrace salt solution 0.1 ml Demineralized water Ad. 1000 ml Trace saltsolution EDTA, Na₂ * 2H₂O 158 mg Na₂MoO₄ * 2H₂O 4.7 mg ZnSO₄ * 7H₂O 70mg MnSO₄ * 4H₂O 18 mg FeSO₄ * 7H₂O 16 mg CuSO₄ * 5H₂O 4.7 mg CoSO₄ *6H₂O 5.2 mg Demineralized water Ad. 1000 ml

Example 2 Isolation and Identification of the Microorganisms

The two strains MA32 and MA113 were selected by determining the nitrilehydratase activity of the resting cells in the presence of 2 mMpotassium cyanide.

Properties of MA32:

Cell form Rods Width 0.6-0.8 μm Length 1.5-3.0 μm Motility + Flagellapolar > 1 Gram reaction − Lysis by 3% KOH + Aminopeptidase (Cerny) +Oxidase + Catalase + Growth at 41° C. − Substrate utilization Adipate −Citrate + Malate + Phenylacetate − D-Glucose + Maltose − Mannitol +Arabinose + Mannose + Trehalose + Sorbitol + Erythrol + Citraconate +Inositol + ADH + Urease − Hydrolysis of gelatin + Hydrolysis ofesculin + Levan from sucrose + Denitrification + Lecithinase +Fluorescence + Pyocyanin −

The profile of the cellular fatty acids is typical of Group IPseudomonas

Analysis of a 484 bp-long segment of the 16S rRNA revealed a 100%agreement with the sequence of Pseudomonas marginalis

It was possible, taking account of all the data, to identify MA32 asPseudomonas marginalis.

Properties of MA113:

Cell form Rods Width 0.6-0.8 μm Length 1.5-3.0 μm Motility + Flagellapolar > 1 Gram reaction − Lysis by 3% KOH + Aminopeptidase (Cerny) +Oxidase + Catalase + Growth at 41° C. − Substrate utilization Adipate −Citrate + Malate + Phenylacetate + D-Glucose + Maltose − Mannitol −Arabinose − Mannose − Trehalose − Inositol − β-Alanine +α-Ketoglutarate + Benzylamine + Hippurate + Azelate + D-mandelate +ADH + Urease − Hydrolysis of gelatin − Hydrolysis of esculin − Levanfrom sucrose − Denitrification − Lecithinase − Fluorescence + Pyocyanin−

The profile of the cellular fatty acids is typical of Group IPseudomonas

Analysis of a 476 bp-long segment of the 16S rRNA revealed a 100%agreement with the sequence of Pseudomonas putida

It was possible, taking account of all the data, to identify MA113 asPseudomonas putida.

Example 3 Determination of the Enzymatic Activity

The cells were grown as described in Example 1, removed from the culturemedium by centrifugation and resuspended in standard buffer (50 mMpotassium phosphate buffer of pH 7.5). 50 μl of this cell suspensionwere added to 700 μl of the standard buffer, and the reaction wasstarted by adding 250 μl of a 200 mM solution of the nitrile in standardbuffer. The concentration of the cells in the cell suspension was inthis case such that the nitrile was 5-30% converted after 10 min at 20°C. After 10 min at 20° C., the reaction was stopped by adding 20 μl of50% concentrated phosphoric acid, and the cells were removed bycentrifugation.

HPLC analysis Column Intersil ODS-3V (GL Sciences Inc.) Mobile phaseMixture of 10 mM potassium phosphate buffer of pH 2.3 and acetonitrilein the ratio 85:15 for methionine nitrile, MHA nitrile and acetonecyanohydrin, and 99:1 for all other substrates Flow rate 1 ml/minDetection UV at 200 nm

The activity of one U is defined as the amount of enzyme which converts1 μmol of methacrylonitrile to the amide in one minute. If the acid wasalso produced in addition to the amide, one U was defined as the amountof enzyme which converts 1 μmol of methacrylonitrile into the amide andacid in one minute.

The relative activities of the strains MA32 and MA113 are depicted inFIG. 1 and in FIG. 2.

Example 4 Influence of Cyanide on the Activity of the Nitrile Hydratase

50 μl of a cell suspension prepared in analogy to Example 3 were addedto 700 μl of the standard buffer which comprised 0, 21.4, 53.6 and 107.1mM potassium cyanide (final concentration 0, 20, 50, 100 mm cyanide).The reaction was started by adding 200 μl of a 200 mM solution of thenitrile in standard buffer which in each case had the same cyanideconcentration as the remaining reaction solution. The concentration ofthe cells in the cell suspension was in this case such that the nitrilewas 16% converted in the mixture without cyanide after 10 min at 20° C.After 10 min at 20° C., the reaction was stopped by adding 20 μl of 50%concentrated phosphoric acid, and the conversion was determined inanalogy to Example 2.

The relative activities for the conversion of methacrylonitrile as afunction of the cyanide concentration are shown in FIG. 3 and in FIG. 4.

Example 5 Conversion of Acetone Cyanohydrin with Resting Cells ofPseudomonas marginalis MA32

Pseudomonas marginalis MA32 cells were grown and centrifuged asdescribed in Example 1. An amount of the cells which comprised 1.16 g ofdry biomass was diluted with 50 mM potassium phosphate buffer of pH 8.0to a final volume of 50 ml. In addition, 0.02 mM of2-methyl-1-propaneboronic acid was added to the reaction mixture.Freshly distilled acetone cyanohydrin was added continuously at 4° C.with vigorous stirring at such a rate that the concentration did notexceed 5 g/l at any point during the reaction. The pH was kept constantat 7.5. The reaction was followed by HPLC as described in Example 3.After 140 min, 10.0 g of the nitrile had been completely converted into10.7 g of amide and 1.4 g of acid.

The time course of the reaction achieved with the strain MA113 isdepicted in FIG. 5.

Example 6 Conversion of Crude MHA Nitrile with Resting Cells ofPseudomonas marginalis MA32

Pseudomonas marginalis MA32 cells were grown and centrifuged asdescribed in Example 1. An amount of the cells which comprised 0.34 g ofdry biomass was diluted with 50 mM potassium phosphate buffer of pH 8.0to a final volume of 70 ml. In addition, 0.02 mM2-methyl-1-propaneboronic acid was added to the reaction mixture. Thecrude MHA nitrile was added continuously at 4° C. with vigorous stirringat such a rate that the concentration did not exceed 10 g/l at any pointduring the reaction. The pH was kept constant at 8.0. The reaction wasfollowed by HPLC as described in Example 3. After 510 min, 10.05 g ofthe nitrile had been completely converted into 11.13 g of amide and 0.31g of acid. This corresponds to a final concentration of 139 g of amideper liter.

The MHA nitrile had been prepared directly from3-methylthiopropionaldehyde and a slight excess of hydrocyanic acid. A50 mM solution of this MHA nitrile in water contained 0.5 mM cyanide(Spektroquant®, Merck).

The time course of the reaction achieved with the strain MA32 isdepicted in FIG. 6.

Example 7 Cloning of the Nitrile Hydratase Gene Cluster from Pseudomonasmarginalis MA 32 and Construction of an Expression Vector

The gene cluster of the nitrile hydratase comprising an α subunit, βsubunit and a nitrile hydratase activator protein whose coexpression isessential for the activity of the nitrile hydratase (Nojiri et al.,1999, Journal of Biochemistry, 125: 696-704) was amplified by PCR usingthe primers 1F and 1R which introduced cleavage sites for therestriction enzymes NdeI and HindIII. The PCR product obtained in thisway was ligated into a vector cut with NdeI and HindIII, with theintroduced genes being under the control of the rhamnose promoter. Theexpression vector produced in this way is called pKE31.

The restriction map is to be found in FIG. 7 and the sequence in SEQ IDNO:1.

The expression plasmid was transformed into the strain E. coli DSM 14459which had been deposited at the Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (DSMZ) on Aug. 22, 2001.

Primers:

1F 5′-CTC CAC CAT ATG AGT ACA GCT ACT TCA ACG-3′ 1R 5′-CTT CAT AAG CTTCTA TCT CGG ATC AAA TGG-3′ 1F: SEQ ID NO: 11 1R: SEQ ID NO: 12

The genes are located on the following segments of SEQ ID NO: 1:

Gene of the α subunit: nt 25-609 Gene of the β subunit: nt 650-1312 Geneof the activator protein: nt 1309-2577 

Example 8 Cloning of the Nitrile Hydratase Gene Cluster from Pseudomonasputida MA113

The gene cluster of the nitrile hydratase consisting of α subunit, βsubunit and a nitrile hydratase activator protein whose coexpression isessential for the activity of the nitrile hydratase (Nojiri et al.,1999, Journal of Biochemistry, 125: 696-704) was amplified by PCR usingthe primers 1F and 1R.

The sequence is to be found in SEQ ID NO: 6.

Primers:

2F 5′-ATG ACG GCA ACT TCA ACC CCT GGT G-3′ 2R 5′-TCA GCT CCT GTC GGC AGTCG-3′ 2F: SEQ ID NO: 13 2R: SEQ ID NO: 14

The genes are located on the following segments of SEQ ID NO: 5:

Gene of the α subunit: nt  1-582 Gene of the β subunit: nt 624-1286 Geneof the activator protein: nt 1283-2360 

Example 9 Heterologous Expression of the Nitrile Hydratases fromPseudomonas marginalis MA 32 in E. coli DSM 14459

E. coli DSM 14459 was deposited in connection with DE 101 55 928.

The cells transformed with pKE31 were grown in LB medium (LB broth,Miller, VWR) which contained 2 mM iron(III) citrate and 100 μg/mlampicillin at 37° C. with shaking. After 12-16 hours, an amount of thepreculture was transferred into a main culture such that the latter hadan OD600 of 0.1. The culture medium of the main culture corresponded tothat of the preculture but additionally contained 2 g/l L-rhamnose. Thecells were harvested after cultivation at 30° C. for 22 hours.

Example 10 Determination of the Enzymatic Activities

The culturing of the cells and the determination of the activity werecarried out as described in Example 9 and Example 3.

The cells of the strain E. coli DSM 14459 transformed with the plasmidpKE31 had a specific activity of 17 U/mg of DBM.

Example 11 Determination of the Enzymatic Activities in the Presence of100 mM Potassium Cyanide

The culturing of the cells and the determination of the activities inthe presence of 100 mM potassium cyanide were carried out as describedin Example 9 and Example 4.

The cells of the strain E. coli DSM 14459 transformed with the plasmidpKE31 had a specific activity of 11 U/mg of DBM.

1. A process for the enzymatic preparation of an amide from a nitrile,which comprises contacting a compound comprising a nitrile group with apolypeptide having nitrile hydratase activity thereby converting thenitrile to the corresponding amide, wherein said polypeptide a)comprises the amino acid sequence of SEQ ID NO: 2, 3, or 5, b) isencoded by a polynucleotide comprising the nucleotide sequence of SEQ IDNO: 1 or 4, or c) is encoded by a polynucleotide which hybridizes withthe complementary sequence of the nucleotide sequence of SEQ ID NO: 1 or4 under stringent conditions, where stringent conditions mean washing in5XSSC at a temperature of from 50 to 65° C.; or microorganism whichproduces the polypeptide, or a lysate thereof.
 2. The process as claimedin claim 1, wherein resting cells of the microorganism are employed. 3.The process as claimed in claim 1, wherein the polypeptide is purified.4. The process as claimed in claim 1, wherein the polypeptide is derivedfrom microorganisms of the genus Pseudomonas.
 5. The process as claimedin claim 4, wherein the polypeptide is derived from employedmicroorganisms of the species Pseudomonas putida or Pseudomonasmarginalis.
 6. The process as claimed in claim 5, wherein the employedmicroorganisms are deposited under the numbers DSM 16275 and DSM 16276.7. The process as claimed in claim 1, wherein the compound has thegeneral formula (I) or (II)

where X is OH, H, alkyl, or NH₂; R is H, saturated alkyl radical having1 to 12 C atoms, branched or unbranched, optionally NH₂-substituted,unsaturated alkyl radicals having a double bond and 1 to 12 C atoms,branched or unbranched, cycloalkyl groups having 3 to 6 C atoms, oralkylene radicals substituted by alkylthio groups, where alkyl herecorresponds to a C₁ to C₃ radical, and alkylene corresponds to adivalent C₃ to C₈ radical, R′ is H, or an alkyl having 1 to 3 C atoms,R″ is a mono- or binuclear unsaturated ring having 6 to 12 C atoms,optionally substituted by one or two alkyl groups (C₁-C₃), Cl, Br, F, oran alkyl nitrile radical having 1 to 6 C atoms.
 8. The process asclaimed in claim 7, wherein the compound is converted in the presence ofhydrocyanic acid or a salt of hydrocyanic acid.
 9. The process asclaimed in claim 8, wherein the conversion is carried out in thepresence of an initial concentration of more than 0.5 mol % cyanide to 3mol % cyanide, based on the compound employed.
 10. The process asclaimed in claim 1, wherein 2-amino-4-methylthiobutyronitrile isemployed as the compound.
 11. The process as claimed in claim 1, wherein2-hydroxy-4-methylthiobutyronitrile is employed as the compound.
 12. Theprocess as claimed in claim 1, wherein 2-hydroxy-2-methylpropionitrileis employed as the compound.
 13. The process as claimed in claim 1,wherein the amide or the solution comprising the amide is separated fromthe microorganism, and the amide is hydrolyzed to the correspondingacid.
 14. The process as claimed in claim 1, wherein the amide or thesolution comprising the amide is separated from the microorganism, andthe amide is hydrolyzed with alkali metal or alkaline earth metalhydroxides to the salts of the corresponding carboxylic acids.
 15. Theprocess as claimed in claim 14, wherein MHA amide is hydrolyzed withcalcium hydroxide, and the calcium salt is obtained.
 16. The process asclaimed in claim 1, wherein a) the microorganism is of the genusPseudomonas and is fermented to obtain the polypeptide or a proteinfraction comprising the polypeptide, and b) transferring the polypeptideor the protein fraction into a medium which comprises the compound. 17.The process as claimed in claim 16, wherein the compound has the generalformula (I) or (II)

where X is OH, H, alkyl, or NH₂; R is H, saturated alkyl radical having1 to 12 C atoms, branched or unbranched, optionally NH₂-substituted,unsaturated alkyl radicals having a double bond and 1 to 12 C atoms,branched or unbranched, cycloalkyl groups having 3 to 6 C atoms, oralkylene radicals substituted by alkylthio groups, where alkyl herecorresponds to a C₁ to C₃ radical, and alkylene corresponds to adivalent C₃ to C₈ radical, R′ is H, or an alkyl having 1 to 3 C atoms,R″ is a mono- or binuclear unsaturated ring having 6 to 12 C atoms,optionally substituted by one or two alkyl groups (C₁-C₃), Cl, Br, F, oran alkyl nitrile radical having 1 to 6 C atoms.